ES2582324T3 - Nanogeles - Google Patents

Abstract

Polymer according to formulas (1) and (2): ** Formula ** where: A is independently selected from a simple direct carbon-carbon bond (10 ie a structure where A is absent), O, N (R1) and S; R1 is independently selected from H and CH3; R2 is independently selected from the group consisting of: (a) C1-C40 alkylene, where the alkylene group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where heteroatoms are independently selected from O, N and S, and / or where the alkylene group is interrupted by one or more groups -SS-; (b) C3-C40 cycloalkylene, where the cycloalkylene group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one or more ring heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where the cycloalkylene group is interrupted by one or more SS-groups outside the ring; (c) C6-C40 arylene, where the arylene group is optionally substituted; (d) C6-C40 heteroarylene, where the heteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the heteroarylene group is optionally substituted; (e) C7-C40 alkylarylene, where the alkylarylene group is optionally substituted and / or where an alkyl part of the alkylarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where heteroatoms are independently selected from O, N and S, and / or where an alkyl part of the alkylarylene group is interrupted by one or more groups -SS-; (f) C7-C40 alkylheteroarylene, where the alkylheteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroarylene group is optionally substituted, and / or where an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where an alkyl part of the alkylheteroarylene group is interrupted by one or several groups -SS-; and (g) a group where two C7-C40 (hetero) arylene groups and / or C7-C40 alkyl (hetero) arylene groups are connected to each other by a group -SS-, where the alkyl part of the (hetero) arylene alkyl group it is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S; R3 is independently selected from the group consisting of: b) C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S; (c) C3-C12 cycloalkyl, where the cycloalkyl group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one, two or three ring heteroatoms, where the heteroatoms are independently selected from O, N and S; (d) C6-C12 aryl, where the aryl group is optionally substituted; (e) C6-C12 heteroaryl, where the heteroaryl group comprises one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S, and where the heteroaryl group is optionally substituted; (f) C7-C14 alkylaryl, where the alkylaryl group is optionally substituted and / or where an alkyl part of the alkylaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms , where the heteroatoms are independently selected from O, N and S; and (g) C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroaryl group is optionally substituted, and / or where an alkyl part of the alkylheteroaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S;

Description

image 1

DESCRIPTION

Nanogeles

5 Field of the invention

[0001] The present invention relates to the design and preparation of polymeric hybrid core-coating nanocarriers whose nuclei are designed to bind therapeutic payload (nucleotides, peptides, proteins, and preferably therapeutic small molecule drugs) and the coating is designed to protect the therapeutic payload, stabilize the nanocarrier, provide biocompatibility to the system, allow specific cells and tissue to be targeted, and provide effective intracellular release of the therapeutic payload of the nanocarrier.

[0002] More in particular, the present invention relates to PA polymers, nanoparticles and nanogels obtained from said PA polymers, wherein said nanogels may comprise a biologically active component.

15 selected from the group of RNA (in particular siRNA or mRNA) or derivatives or fragments thereof, DNA or derivatives or fragments thereof, (oligo) peptides and derivatives thereof, and proteins and derivatives thereof.

Background of the invention

[0003] The present invention is in the field of siRNA delivery (siRNA refers to small interfering RNA that is also known as short interfering RNA or silencing RNA) and mRNA delivery (mRNA refers to micro RNA) .

[0004] RNA interference (RNAi) represents a promising strategy to affect gene expression

25 pathogens due to their ability to silence gene expression in a sequence specific way. The selection of different siRNA sequences resulted in the identification of different potential therapeutic oligonucleotides for different therapeutic uses. However, the delivery of ARNsi is a big problem. Naked siRNA is rapidly degraded by nucleases in the biological fluid (the half-life is of the order of minutes), and a carrier is generally required that provides sufficient protection against degradation in the extracellular environment and ensures the effective crossing of cell barriers during intracellular absorption and treatment. In particular, the highly negative charged siRNA can pass cell membranes only with great difficulty. In addition, once in the intracellular fluid, efficient unpacking of siRNA from the carrier is required to show its therapeutic activity. As a consequence, several polymeric non-viral carriers for the efficient delivery of RNA, DNA and siRNA have been investigated.

[0005] US 2002/0131951 (now US 6,998,115), US 2004/0071654 (now US 7,427,394), US 2005/0244504 and US 2010/0036084, all incorporated by reference, reveal poly (amino polymers) ester) obtained from bisacrylamides and functionalized primary amines. In their cationic form, poly (amino ester) polymers complex with DNA molecules or fragments thereof.

[0006] US 2008/0242626, incorporated by reference, discloses poly (amino ester) polymers based on bisacrylamides and functionalized primary amines, where poly (amino ester polymers) undergo an end modification. Where the poly (amino ester) polymer is aminosubstituted, the poly (amino ester) polymer is reacted with an electrophile. Where the poly (amino ester) polymer is acrylate substituted, the poly (amino) polymer

Ester) is reacted with a nucleophile. These modified poly (amino ester) polymers at the end are used for the delivery of DNA and RNA.

[0007] WO 2010/065660, incorporated by reference, discloses biodegradable polydulfide amines and complexes thereof with for example RNA, DNA, siRNA and other oligonucleotides.

[0008] US 2010/0041739, incorporated by reference, discloses polyalkylene imine polymers that can be used for delivery of eg DNA.

[0009] US 2010/0028445, incorporated by reference, discloses polyamine amine polymers comprising

55 pendant disulfide groups, pendant thiol groups or pendant activated thiol groups and a method for the preparation of such poly (amino amine) polymers, where bisacryloyl monomers are reacted with a primary amine and / or a secondary diamine where one of the amines contains a disulfide group. Poly (amino amine) polymers can be converted into hydrogels forming disulfide groups between the poly (amino amine) chains.

[0010] C. Lin et al., Bioconjug. Chem. 18, 138-145, 2007, incorporated by reference, discloses poly (amido amine) s containing disulfide bonds that are prepared by a Michael addition of primary amines and N, N'-cystamine bisacrylamide containing disulfide.

[0011] L.J. van der Aa et al., J. Control. Release 148, e85, 2010, incorporated by reference, discloses poly (amido) s 65 containing disulfide bonds that are prepared by a Michael addition of primary amino alcohols, N, N'cistamine bisacrylamide containing disulfide and 1,2-diaminoethane .

image2

[0012] R. Namgung et al., Biotechnol. Lett. 32, 755, 2010, incorporated by reference, discloses poly (amido amine) s containing disulfide bonds that are prepared by a Michael addition of 4-amino-1-butanol and N, N'-cystamine bisacrylamide containing disulfide.

5 [0013] P. Vader et al., Pharm. Res. 28, 1013, 2011, incorporated by reference, discloses poly (amido amine) s containing disulfide bonds that are prepared by a Michael addition of 4-amino-1-butanol, N, N'-cystamine bisacrylamide containing disulfide and 1,2-daminoethane.

[0014] D. Vercauteren et al., Biomaterials 32, 3072, 2011, incorporated by reference, discloses poly (amido amine) s containing disulfide bonds that are prepared by a Michael addition of 4-amino-1-butanol and N, N'-cystamine bisacrylamide containing disulfide.

[0015] Cationic polymer systems known in the state of the art still have several disadvantages. In

In particular, the need for the presence of excess polymer (cytotoxic) in the therapeutic formulation, frequently low handling and storage stability of therapeutically charged nanogels, as well as limited efficiencies in endosomal leakage and cytosolic unpacking of the therapeutic payload (nucleotide) from the complex are still important challenges in this field.

20 Summary of the invention

[0016] The present invention relates to a PA polymer (PolyAcril) according to the general formulas (1) and (2):

image3

where:

A is independently selected from a simple direct carbon-carbon bond (ie, a structure where A is absent), O, N (R1) and S;

R1 is independently selected from H and CH3;

R2 is independently selected from the group consisting of:

(A) C1-C40 alkylene, where the alkylene group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where the alkylene group is interrupted by one or more SS- groups; (b) C3-C40 cycloalkylene, where the cycloalkylene group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one or more ring heteroatoms, where the heteroatoms are independently selected from O, N and S , and / or where the cycloalkylene group is interrupted by one or more SS-outside ring groups;

image4

5 (c) C6-C40 arylene, where the arylene group is optionally substituted;

(d)

C6-C40 heteroarylene, where the heteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the heteroarylene group is optionally substituted;

(and)

C7-C40 alkylarylene where the alkylarylene group is optionally substituted and / or where a part of

The alkyl of the alkylarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where an alkyl part of the alkylarylene group is interrupted by one

or several S-S- groups;

(F)

C7-C40 alkylheteroarylene, where the alkylheteroarylene group comprises one, two or three heteroatoms

15 independently selected from O, N and S and / or where the alkylheteroarylene group is optionally substituted, and / or where an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where an alkyl part of the alkylheteroarylene group is interrupted by one or more -SS groups; Y

20 (g) a group where two C7-C40 (hetero) arylene groups and / or C7-C40 alkyl (hetero) arylene groups are connected to each other by an SS- group, where the alkyl part of the (hetero) arylene alkyl group it is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S;

R3 is independently selected from the group consisting of:

(to)

H;

(b)

C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally substituted and / or is

optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S;

(C)

C3-C12 cycloalkyl, where the cycloalkyl group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one, two or three ring heteroatoms, where the heteroatoms are independently selected from O, N and S;

(d)

C6-C12 aryl, where the aryl group is optionally substituted;

(E) C6-C12 heteroaryl, where the heteroaryl group comprises one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S, and where the heteroaryl group is optionally substituted;

(f) C7-C14 alkylaryl, where the alkylaryl group is optionally substituted and / or where an alkyl part of the alkylaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally

Interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S; Y

(g) C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroaryl group is optionally substituted, and / or where an alkyl part of the alkylheteroaryl group is linear or branched and is optionally

(Partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S;

R4 is independently selected from the group consisting of:

50 (a) H;

(b)

C1-C10 alkyl, where the alkyl group can be linear or branched;

(C)

C3-C12 cycloalkyl;

(d)

C6-C14 aryl;

(e) C6-C14 heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S;

(F)

C7-C14 alkylaryl; Y

(g)

C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S;

60 R5 is independently selected from the group consisting of:

(to)

C1-C12 alkylene, where the alkylene group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S:

(b)

C3-C12 cycloalkylene, where the cycloalkylene group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one or more ring heteroatoms, where the heteroatoms are independently selected from O, N and S;

(C)

C6-C12 arylene, where the arylene group is optionally substituted;

image5

5 (d) C6-C12 heteroarylene, where the heteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the heteroarylene group is optionally substituted;

(e) C7-C12 alkylarylene, where the alkylarylene group is optionally substituted and / or where an alkyl part of the alkylarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where heteroatoms are independently

10 selected from O, N and S; Y

(f) C7-C12 alkylheteroarylene, where the alkylheteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroarylene group is optionally substituted, and / or where an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms,

Where the heteroatoms are independently selected from O, N and S; POL represents a polymer core having a weight average molecular weight Pm of about 300 to about 25,000;

a = 2-64; and 20 b = 1 -50.

[0017] The present invention further relates to a nanoparticle according to the general formulas (3) and (4):

image6

where R1, R2, R3, R4, R5, A, POL, a and b are as defined above, and where R6 is selected from the group consisting of:

30 (a) C1-C40 alkylene, where the alkylene group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where the alkylene group is interrupted by one or more SS- groups; or

(b) C3-C40 cycloalkylene, where the cycloalkylene group is optionally substituted and / or is optionally

(Partially) unsaturated and / or optionally comprises one or more ring heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where the cycloalkylene group is interrupted by one or more SS-outside groups of the ring ; Y

FG is a functional group.

[0018] The present invention further relates to a nanogel comprising a PA polymer (PolyAcryl) according to general formulas (1) or (2), where the nanogel is formed by crosslinking the PA polymer.

image7

[0019] The present invention further relates to nanogels having a modified surface.

[0020] The present invention further relates to nanogels comprising a biologically active component.

[0021] The present invention further relates to the use of nanogels and the use of nanogels in the delivery of a biologically active component to a mammal.

[0022] The present invention further relates to pharmaceutical compositions comprising the nanogel and a pharmaceutically acceptable carrier.

[0023] The present invention further relates to a method of delivering a biologically active component to a mammal, where a nanogel or a composition comprising such a nanogel is administered to a mammal.

Description of the figures 15 [0024]

Figure 1 shows the ability of PA polymers to bind siRNA as set forth in an EtBr (ethidium bromide) assay. A low fluorescence signal is indicative of effective shielding (protection) of siRNA. Figure 2 shows the ability of nanogels comprising a PA polymer to bind and shield RNAi as set forth in an EtBr (ethidium bromide) assay. Figure 3 shows that nanogels have a higher ability to bind and shield RNAi than PA polymers (compare samples no .: 12 vs. 4 and 13 vs. 3 in higher polymer / siRNA ratio). Figure 4 shows the gene that silences the efficiency in H1299 cells of PA polymers. Dark gray bars

25 show the percentage of luciferase expression. Light gray bars are controls with insensitive siRNA. Figure 5 shows the gene that silences the efficiency in H1299 cells of PA polymers. Figure 6 shows the gene that silences the efficiency of some nanogels with different core composition. Figure 7 shows cell viability curves of H1299 cells for a PA polymer (sample No. 4) and a nanogel (sample No. 12) as a concentration function. Figure 8 shows H1299 luciferase silencing under serum conditions by siRNA-loaded nanogels in different polymer / siRNA ratios (w / w) and different doses of siRNA (ng per 100 µL of transfection medium). Figure 9 shows luciferase silencing of H1299 cells under serum conditions by siRNA-loaded nanogels (siRNA siRN 72) in different polymer / siRNA mix ratios.

Figure 10 shows silencing of luciferase from H1299 cells by a nanogel loaded with siRNA (sample No. 12) having its surface modified with amount of PEG (% by weight with respect to the polymer of the nanogel). Figure 11 shows silencing of luciferase from H1299 cells by a nanogel loaded with siRNA (sample No. 12), using a freshly prepared siRNA-nanogel solution, a frozen and thawed siRNA-nanogel solution and a lyophilized siRNA nanogel solution, respectively. The black bar gives the expression of luciferase (set at 100%) of the control experiments, using antisense coding siRNA. It is shown that the three differently treated formulations with antisense siRNA show equal luciferase silencing activity.

Detailed description of the invention

[0025] The verb "to understand", as used in this description and in the claims and their conjugations, is used in its non-limiting sense to say that the elements that follow the word are included, but the elements that are not They specifically mention they are not excluded. In addition, the reference to an element by the indefinite article "a" or "a" does not exclude the possibility that there is more than one of said element, unless the context clearly specifies that there is one and only one of the elements. The indefinite article "a" or "such" normally means "at least one".

[0026] The term "siRNA" also encompasses "mRNA" (micro RNA).

55 PA polymer

[0027] According to the present invention, it is preferred that the polymer according to formulas (1) and (2) have an average molecular weight in weight Pm in the range of about 10,000 to about 1,000,000, more preferably in the range of about 20,000 to approximately 500,000 g / mol.

[0028] According to a preferred embodiment, A is N (R1) or O, most preferably N (R1).

[0029] According to another preferred embodiment of the present invention, R1 is H.

[0030] According to another preferred embodiment, R2 is selected from: (a1) C1-C20 alkylene, preferably C2-C12 alkylene, where the alkylene group can be linear or branched and is optionally substituted and / or optionally (partially ) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where the alkylene group is interrupted by one or more SS- groups;

image8

5 (b1) C3-C20 cycloalkylene, preferably C5-C12 cycloalkylene, where the cycloalkylene group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises 1, 2 or 3 ring heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where the cycloalkylene group is interrupted by one or more SS-outside ring groups; (c1) C6-C20 arylene, preferably C6-C12 arylene, where the arylene group is optionally substituted;

10 (d1) C6-C20 heteroarylene, preferably C6-C12 heteroarylene, wherein the heteroarylene group comprises 1, 2 or 3 heteroatoms independently selected from O, N and S and / or where the heteroarylene group is optionally substituted; (e1) C7-C20 alkylarylene, preferably C7-C12 alkylarylene where the alkylarylene group is optionally substituted and / or where an alkyl part of the alkylarylene group is linear or branched and is optionally

15 (partially) unsaturated and / or is optionally interrupted by 1, 2 or 3 heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where an alkyl part of the alkylarylene group is interrupted by one or more groups H.H-; (f1) C7-C20 alkylheteroarylene, preferably C7-C12 alkylheteroarylene, wherein the alkylheteroarylene group comprises 1-3 heteroatoms independently selected from O, N and S and / or where the group

Alkylheteroarylene is optionally substituted, and / or where an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where an alkyl part of the alkylheteroarylene group is interrupted by one or more SS- groups; and (g1) a group where two C7-C20 (hetero) arylene groups and / or C7-C20 alkyl (hetero) arylene groups, preferably

Two C7-C12 (hetero) arylene groups and / or C7-C12 alkyl (hetero) arylene groups are connected to each other by an SS- group, where the alkyl part of the alkyl (hetero) arylene group is linear or branched and it is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S.

[0031] According to another preferred embodiment of the present invention, R2 is selected from group (a1) or group (b1), more preferably group from group (a1), and in particular from group (a1) where the alkylene group C1-C40 is interrupted by one or more groups -SS.

[0032] According to a preferred embodiment, when R3 is substituted C1-C10 alkyl, the alkyl group is substituted by a group selected from -OH, -OR7, -NH2; -NH (R7), -N (R7) 2, -C (O) OR7, -C (O) R7, -C (O) NHR7 and -C (O) NR72, where R7 is independently selected from the group consisting in:

(to)

H;

(b)

C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally substituted and / or is

Optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S;

(c) C3-C12 cycloalkyl, where the cycloalkyl group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one, two or three ring heteroatoms, where the heteroatoms are independently selected from O, N and S;

(D) C6-C12 aryl, where the aryl group is optionally substituted;

(and)

C6-C12 heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the heteroaryl group is optionally substituted;

(F)

C7-C14 alkylaryl, where the alkylaryl group is optionally substituted and / or where an alkyl part of the alkylaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally

50 interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S; Y

(g) C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroaryl group is optionally substituted, and / or where an alkyl part of the alkylheteroaryl group is linear or branched and is optionally (partially)

55 unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S.

[0033] According to a preferred embodiment, when R3 is substituted C3-C12 cycloalkyl, the cycloalkyl group has a pendant group selected from -OH, -OR7, -NH2; -NH (R7), - N (R7) 2, -C (O) OR7, -C (O) R7, -C (O) NHR7 and -C (O) NR72, 60 where R7 is as it has been defined above.

[0034] According to a preferred embodiment, when R 3 is C 6 -C 12 aryl, C 6 -C 12 heteroaryl, C 7 -C 14 alkylaryl or substituted C 7 -C 14 alkylheteroaryl, the alkylaryl or alkyl heteroaryl group is substituted with, preferably with one, two

or three of -OH, -OR7, -NH2; -NH (R7), -N (R7) 2, -C (O) OR7, -C (O) R7, -C (O) NHR7 and -C (O) NR72, where R7 is as it has been defined above.

image9

[0035] In the PA polymer according to the present invention which is represented by the general formulas (1) and (2), the polymer polymer core is preferably based or selected from a branched, hyperbranched, multibrack, dentritic (co) polymer star type, said (co) polymer preferably has 2-64, more preferably 2-32, even more preferably 2-16 terminal amino groups, preferably primary amino groups. Therefore, a is 2-64, preferably 2-32 and in particular 2-16. The (co) polymers may comprise different linear or branched spacers comprising one or more heteroatoms selected from the group consisting of O, N and S, preferably O and N.

[0036] According to the present invention, b is 1-50, preferably 1-30 and in particular 1-10.

[0037] It is well known in the art that dendritic (co) polymers are not always perfectly branched and may therefore have a hyperbranched structure. The degree of branching (DB) can be defined by:

image10

15 where D is the number of dendritic, L the number of linear and T the number of terminal units. Perfect dendrimers will have a DB of 1, while hyperbranched (co) polymers typically have a DB of 0.4 to 0.5 to even 0.9. In this patent application, the term "dendrimer" should be understood to include "perfectly branched dendrimers" as well as "imperfectly branched dendrimers" which are also referred to as "(co) hyperbranched polymers." Alternatively, the term "(co) hyperbranched polymers" may also comprise (co) "real" hyperbranched polymers. That is, these macromolecules are deliberately prepared to have a hyperbranched structure. The term "dendrimer" should be understood as comprising both dendrimeric homopolymers and dendrimeric copolymers. The term "copolymer" includes polymers formed by at least two different monomers.

[0038] Hyperbranched polymers can be obtained from random polymerization of monomers in the presence of at least one polyfunctional monomer capable of introducing branching. Such a synthetic scheme is shown by Hawker and Devonport in "Step-Growth Polymers for High-Performance Materials: New Synthetic Methods," Hedrick, JL and Labadie, JW, Eds., Am. Chem. Soc., Washington, DC, 1996, pp. 191-193. Hult et al., In "Advances in Polymer Science," Vol. 143 (1999), Roovers, J., Ed., Springer, New York, pp. 1-34, present a review of hyperbranched polymers.

[0039] Highly branched dendritic polymers are discussed for example in "Polymeric Materials Encyclopedia", Vol. 5 (1996), J.C. Salamone, Ed., CRC Press, New York, pp. 3049-3053. Dendritic polymers have

35 generally a non-linear architecture and are intrinsically globular in shape. Synthesis methods by discrete stages are used to prepare dendrimers or highly branched pure compounds. As Hawker and Devonport mention in "Step-Growth Polymers for High-Performance Materials: New Synthetic Methods", Hedrick, J. L. and Labadie, J. W., Eds., Am. Chem. Soc., Washington, D. C., 1996, pp. 186-196, if the macromolecule has highly regular branching that follows a strict geometric pattern, it is a dendrimer. Dendrimers are typically monodisperse and are prepared in a multiphase method with purifications at each stage. The architecture of dendrimers is also discussed by Roovers and Comanita in "Advances in Polymer Science", Vol. 142 (1999), Roovers, J., Ed., Springer, New York, pp. 179-228. Dendrimers consist of a core molecule that defines the center of symmetry of the molecule, and branching strata. Tomalia et al., Angew. Chem. Int. Ed. Eng., 29 (1990), 138-175 disclose "starburst" dendrimers consisting of an initiator nucleus and branching groups.

[0040] Preferably, the POL polymer core is based on PEI (commercially available from eg Denditrech, Inc.), Astramol® polymers (DSM), JEFFAMINE® polymers (Huntsman), PAMAM polymers (sometimes also called PANAM polymers) , PPI polymers, PEAN polymers and PEAC polymers. The term "PEI" refers to polyethyleneimines. The term "PAMAM" refers to poly (amide amine) polymers that are commercially available under the trade name Starburst®. The term "PPI" refers to polymers of polypropylene imine. The term "PEAN" refers to poly (polyamine ester) polymers. The term "PEAC" refers to poly (ether amine) polymers. All these polymers are known in the art. Therefore, it is preferred that the POL polymer core be based on or selected from the group consisting of PEI, PAMAM, PPI, PEAN and PEAC.

[0041] According to the present invention, it is preferred that the weight average molecular weight Pm of the polymer core POL be from about 300 to about 5,000, more preferably from about 600 to about 5,000.

[0042] Preferred polymers used for the POL polymer core are polymers (polyether amines) of the JEFFAMINE® T series that are commercially available with an average molecular weight in weight Pm in the range of about 440 to about 5,000. Suitable types of JEFFAMINE® T polymers include T-403 (Pm = 440) and T-3000 (Pm = 3,000).

image11

[0043] Another group of preferred polymers that can be used for the polymer core POL are those of PEI. PEI can have either a linear or branched structure. Linear PEI is commercially available (jetPEI, Polyplus-Transfection Co .; Exgen 500, Fermentas Co.) and is normally prepared by hydrolysis of poly (2-ethyl-2-oxazoline. Those of branched PEI are obtained from aziridine and these Polymers have a highly branched structure and comprise about 25% of primary amine groups, about 50% secondary amine groups, and about 25% tertiary amine groups Preferably, PEI has a weight average molecular weight Mn of about 600 to about 3,000, more preferably from about 600 to about

2,000 Those of linear PEI can be represented by the general formula (5a):

image12

[0044] Those of branched PEI can be represented by the general formula (5b):

image13

where n is so that the PEI has a weight average molecular weight Mn of about 300 to

About 5,000, more preferably about 300 to about 3,000, even more preferably about 600 to about 2,500. Such of PEI are for example available from Sigma-Aldrich.

[0045] Another group of preferred polymers used for the POL polymer core are polyamine amine polymers.

20 hyperbranched and dendrimers, preferably those of the first and fourth generation, more preferably those of the first and third generation. These polymers are commercially available from Dentritech, Inc., and have a weight average molecular weight Pm in the range of about 1,400 (first generation) to about

15,000 (fourth generation).

[0046] Another group of preferred polymers used for the POL polymer core are the polymers represented by the general formulas (6) - (9):

N (R8) 3-n [(CR92) m-N (R10R11) n (6) 30 [(R10R11) N- (CR92) m] 2N-P-N [(CR92) m-N (R10OR11)] 2 (7)

N (R8) 3-n [(CR92) m-C (O) N (R9) - (CR92) m-N (R12R13)] n (8)

35 [(R12R13) N- (CR92) m-N (R9) C (O) - (CR92) m] 2N-P-N [(CR92) -C (O) NH- (CR92) m-N (R12R13)] 2 (9)

image14

where:

R8 is a hydrogen atom, a linear or branched C1-C20 alkyl group or a group - [(CR142) q-X] or-R15, where X is O or N (R8); 5 m is 2.3o4; n is 2 or 3;

o is 1-10; q is 2, 3 or 4; P is - (CR92) m-, a C6-C12 arylene group, a C6-C12 cycloalkylene group or a group - [(CR142) q-X] p-C (R14) 2] -, where

10 X isOoN (R8) andpes1-10; R9 is a hydrogen atom or a linear or branched C1-C6 alkyl group; R10 and R11 are independently a hydrogen atom, a linear or branched C1-C6 alkyl group or a group of the formula - (CR142) qNR16R17, provided that R10 and R11 are not both a linear or branched C1-C6 alkyl group; R16 and R17 are independently a hydrogen atom, a linear or branched C1-C6 alkyl group or a group

15 of the formula - (CR142) qNR18R19, provided that R16 and R17 are not both a linear or branched C1-C6 alkyl group; R18 and R19 are independently a hydrogen atom, a linear or branched C1-C6 alkyl group or a group of the formula - (CR142) qNR20R21, provided that R18 and R19 are not both a linear or branched C1-C6 alkyl group; R20 and R21 are independently a hydrogen atom or a linear or branched C1-C6 alkyl group, provided that R20 and R21 are not both a linear or branched C1-C6 alkyl group;

R22 is a hydrogen atom or a methyl group, provided that at least one R22 is a hydrogen atom; R15 is a hydrogen or a linear or branched C1-C20 alkyl group or a group - [(CR142) q-X] or-R15 as defined above; R12 and R13 are independently a hydrogen atom, a linear or branched C1-C6 alkyl group or a group of the formula - (CR92) m-C (O) NH- (CR92) m-N (R23R24), provided that R12 and

R13 are not both a linear or branched C1-C6 alkyl group; R23 and R24 are independently a hydrogen atom, a linear or branched C1-C6 alkyl group or a group of the formula - (CR92) mC (O) NH- (CR92) mN (R25R26), provided that R23 and R24 are not both a linear or branched C1-C6 alkyl group; R25 and R26 are independently a hydrogen atom, a linear or branched C1-C6 alkyl group or a group of

The formula - (CR92) m-C (O) NH- (CR92) m-N (R27R28), provided that R25 and R26 are not both a linear or branched C1-C6 alkyl group; R27 and R28 are independently a hydrogen atom or a linear or branched C1-C6 alkyl group, provided that R27 and R28 are not both a linear or branched C1-C6 alkyl group.

[0047] In formulas (6) - (9), preferred group polymers used for the polymer core POL is the group where:

R8 and R15 is a hydrogen atom or a group - [(CR142) q-X] or-R15, where X is NH;

R9, R20, R21 and R21 are a hydrogen atom; 2) qNR16R17

R10 and R11 are independently a hydrogen atom or a group of the formula - (CR14; 2) qNR18R19

R16 and R17 are independently a hydrogen atom or a group of the formula - (CR14; 2) qNR20R21

R18 and R19 are independently a hydrogen atom or a group of the formula - (CR14; R12 and R13 are independently a hydrogen atom or a group of the formula - (CR92) mC (O) NH- (CR92) mN (R23R24 );

R23 and R24 are independently a hydrogen atom or a group of the formula - (CR92) m-C (O) NH- (CR92) mN (R25R26); R25 and R26 are independently a hydrogen atom or a group of the formula - (CR92) m-C (O) NH- (CR92) mN (R27R28); and R27 and R28 are independently a hydrogen atom.

[0048] In formulas (6) - (9), it is also preferred that m and q be 2:

[0049] A more preferred class of PA polymers according to the present invention can be represented by the general formulas (10) and (11):

image15

R1 is independently selected from H and CH3; Y is O or N (R); r is 2, 3 or 4; Z is S-S-;

10 R29 is independently selected from the group consisting of:

(to)

H;

(b)

C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally substituted and / or is

optionally (partially) unsaturated and / or is optionally interrupted by 1, 2 or 3 heteroatoms, where the heteroatoms are independently selected from O, N and S;

(C)

C3-C12 cycloalkyl, where the cycloalkyl group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises 1, 2 or 3 ring heteroatoms, where the heteroatoms are independently selected from O, N and S;

(d)

C6-C12 aryl, where the aryl group is optionally substituted;

(E) C6-C12 heteroaryl, wherein the heteroaryl group comprises 1, 2 or 3 heteroatoms independently selected from O, N and S and / or where the heteroaryl group is optionally substituted;

(f) C7-C14 alkylaryl, where the alkylaryl group is optionally substituted and / or where an alkyl part of the alkylaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by 1, 2 or 3 heteroatoms , where heteroatoms are independently selected from

25 O, N and S; and

(g) C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises 1, 2 or 3 heteroatoms independently selected from O, N and S and / or where the alkylheteroaryl group is optionally substituted, and / or where an alkyl part of the alkylheteroaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by 1, 2 or 3 heteroatoms, where

30 heteroatoms are independently selected from O, N and S; the alkylheteroaryl group is optionally substituted, preferably with 1, 2 or 3 of -OH, -OR29, -NH2 -NH (R29) or -N (R29) 2;

R30 is independently selected from the group consisting of:

35 (a) H;

(b)

C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S;

(C)

C3-C12 cycloalkyl, where the cycloalkyl group is optionally substituted and / or is optionally

(Partially) unsaturated and / or optionally comprises one, two or three ring heteroatoms, where the heteroatoms are independently selected from O, N and S;

(d)

C6-C12 aryl, where the aryl group is optionally substituted;

(and)

C6-C12 heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the heteroaryl group is optionally substituted;

(F)

C7-C14 alkylaryl, where the alkylaryl group is optionally substituted and / or where an alkyl part of the alkylaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally

image16

5 interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S; Y

(g) C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroaryl group is optionally substituted, and / or where an alkyl part of the alkylheteroaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S;

R31 is independently selected from the group consisting of:

15 (a) H;

(b)

C1-C10 alkyl, where the alkyl group can be linear or branched;

(C)

C3-C12 cycloalkyl;

(d)

C6-C12 aryl;

(and)

C6-C12 heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S;

(F)

C7-C14 alkylaryl; Y

(g)

C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S;

R32 is independently selected from the group consisting of:

(to)

C1-C12 alkylene, where the alkylene group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S;

(b)

C3-C12 cycloalkylene, where the cycloalkylene group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one or more ring heteroatoms, where the heteroatoms are independently selected from O, N and S;

(C)

C6-C12 arylene, where the arylene group is optionally substituted;

(d) C6-C12 heteroarylene, where the heteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the heteroarylene group is optionally substituted;

(and)

C7-C14 alkylarylene, where the alkylarylene group is optionally substituted and / or where an alkyl part of the alkylarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S; Y

(F)

C7-C14 alkylheteroarylene, where the alkylheteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroarylene group is optionally substituted, and / or where an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S; Y

45 a = 2-64; and b = 1 to 50.

[0050] According to a preferred embodiment, when R30 is substituted C1-C10 alkyl, the alkyl group is substituted by a group selected from -OH, -OR7, -NH2; -NH (R7), -N (R7) 2, -C (O) OR7, -C (O) R7, -C (O) NHR7 and -C (O) NR72, where R7 is independently selected from the group consisting in:

(to)

H;

(b)

C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally substituted and / or is

Optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S;

(C)

C3-C12 cycloalkyl, where the cycloalkyl group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one, two or three ring heteroatoms, where the heteroatoms are independently selected from O, N and S;

(d)

C6-C12 aryl, where the aryl group is optionally substituted;

(and)

C6-C12 heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the heteroaryl group is optionally substituted;

(F)

C7-C14 alkylaryl, where the alkylaryl group is optionally substituted and / or where an alkyl part of the alkylaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally

65 interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S; Y

image17

(g) C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroaryl group is optionally substituted, and / or where an alkyl part of the alkylheteroaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are

5 independently selected from O, N and S.

[0051] According to a preferred embodiment, when R30 is substituted C3-C12 cycloalkyl, the cycloalkyl group has a pendant group selected from -OH, -OR7, -NH2; -NH (R7), - N (R7) 2, -C (O) OR7, -C (O) R7, -C (O) NHR7 and -C (O) NR72, where R7 is as defined previously.

[0052] According to a preferred embodiment, when R30 is C6-C12 aryl, C6-C12 heteroaryl, C7-C14 alkylaryl or substituted C7-C14 alkylheteroaryl, the alkylaryl or alkylheteroaryl group is substituted with, preferably with one, two

or three of -OH, -OR7, -NH2; -NH (R7), -N (R7) 2, -C (O) OR7, -C (O) R7, -C (O) NHR7 and -C (O) NR72, where R7 is as defined previously.

[0053] In a more preferred class of PA polymers according to general formulas (10) and (11):

R1 is independently selected from H and CH3; Y is N (R1); r is 2; Z is S-S-; R29 is independently selected from H and C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S;

R30 is independently selected from H and C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where heteroatoms are independently selected from O, N and S; R31 is independently selected from H and C1-C10 alkyl, where the alkyl group can be linear or branched; and R32 is C1-C10 alkylene, where the alkylene group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected of O, N and S.

[0054] In an even more preferred class of PA polymers according to general formulas (10) and (11):

R1 is H; Y is N (R1); r is 2; Z is S-S-; R29 is H; R30 is independently selected from H and C1-C10 alkyl, where the alkyl group is linear and is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S; R31 is H; Y

R32 is C1-C10 alkylene, where the alkylene group can be linear or branched.

[0055] Still in an even more preferred class of PA polymers according to general formulas (10) and (11):

R1 is H; Y is N (R1); r is 2; Z is S-S-; R29 is H;

R30

it is C1-C10 alkyl, where the alkyl group is linear and is optionally interrupted by one, two or three

55 heteroatoms, where the heteroatoms are independently selected from O, N and S; R31 is H; and R32 is C1-C10 alkylene, where the alkyl group can be linear or branched.

[0056] In a more preferred class of PA polymers according to general formulas (10) and (11):

Y is NH; r is 2; Z is S-S-; R29 is H;

R30 is C1-C6 alkyl; R31 is H; and R32 is C1-C10 alkylene, where the alkylene group is linear.

image18

[0057] In all of these preferred classes of PA polymers according to formulas (10) and (11), it is preferred that when R30 is substituted C1-C10 alkyl, the alkyl group is replaced by a group selected from -OH, -OR7 , -NH2; -NH (R7),

5 N (R7) 2, -C (O) OR7, -C (O) R7, -C (O) NHR7, and -C (O) NR72, where R7 is as defined above. Preferably, the alkyl group is substituted by -OH, -NH2, -NH (R7), or -N (R7) 2; more preferably by -OH or substituted -NH2.

PA polymer preparation process

[0058] According to an embodiment of the present invention, the PA polymers according to the present invention as represented by the general formulas (1) and (2) can be prepared by a process comprising the steps of:

(1) react a monomer (I) according to the general formula (12):

image19

where R1, A and R2 are as defined above, with a monomer (II) according to the general formula (13)

or a monomer (III) according to formula (14):

20 (H2N) -R3 (13)

HR3N-R5-NR3H (14)

Where R3 and R5 are as defined above, in a molar ratio of monomer (I): monomer

(II) from about 1.5: 1 to about 10: 1 to form a macromer according to the general formulas (15) or (16):

image20

where b is as defined above; Y

(2) reacting the macromer according to the general formulas (15) or (16) with a polymer according to the formula (17); [(R42) N] a-POL (17)

where a, POL and R4 are as defined above and where at least one R4 is H.

[0059] Therefore, in this reaction, a group -C (O) -C (R1) = C reacts with a group (R42) N of POL under the formation 5 of a fraction -C (O) -CH ( R1) -CH2-N (R4) - (Michael's addition) as will be clear to one skilled in the art.

[0060] Accordingly, the present invention also relates to the PA polymer according to the general formulas (1) and (2) that can be obtained by this process.

[0061] According to a preferred embodiment of the present invention, the PA polymers according to the present invention as represented by the general formulas (10) and (11) can also be prepared by this process comprising the steps of:

(1) react a monomer (III) according to the general formula (18):

image21

where R1, Y, R29 and r are as defined above, with a monomer (IV) according to the general formula

(19) or a monomer (V) according to formula (20):

20 (H2N) -R30 (19)

HR30N-R32-NR30H (20)

Where R30 and R32 are as defined above, in a molar ratio of monomer (I): monomer

(II) from about 1.5: 1 to about 10: 1 to form a macromer according to the general formulas (21) or (22):

image22

where b is as defined above; Y

(2) reacting the macromer according to the general formulas (21) or (22) with a polymer according to the formula (23); [(R312) N] a-POL (23)

image23

where a, POL and R31 are as defined above and where at least one R31 is H.

[0062] According to the present invention, it is preferred that step (1) of the process be carried out at a temperature ranging from room temperature to about 100 ° C, preferably from about 30 ° to about 80 ° C.

[0063] According to the present invention, it is preferred that step (2) of the process be carried out at a temperature ranging from room temperature to about 100 ° C, preferably from about 30 ° to about 80 ° C.

10 The nanoparticle

[0064] The present invention also relates to a nanoparticle. The nanoparticle according to the present invention is represented by the general formulas (3) and (4):

fifteen

image24

where R1, R2, R3, R4, R5, R6, A, POL, FG, a and b are as described above.

[0065] According to a preferred embodiment, the nanoparticle is represented by the general formulas (24) and (25):

image25

image26

where R1, R6, R29, R30, R31, R32, Y, Z, POL, FG, a, b and r as defined above. The FG functional group is a substituent that is capable of forming a covalent bond with a complementary functional group (CFG) of a reagent for post-modification. The FG group is preferably selected from functional groups that allow the formation of a covalent bond with a CFG group, preferably under biocompatible reaction conditions, in

Particularly under conditions of physiological pH and ambient temperature and in aqueous systems. Such FG groups are well known to the person skilled in the art. In particular, the group FG is selected from the group consisting of a group selected from -OH, -OR7, -NH2; -NH (R7), -N (R7) 2, -C (O) OR7, -C (O) R7, -C (O) NHR7 and -C (O) NR72, where R7 is independently selected from the group consisting in:

(a ") H; (b") C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms , where the heteroatoms are independently selected from O, N and S; (c ") C3-C12 cycloalkyl, where the cycloalkyl group is optionally substituted and / or is optionally

(Partially) unsaturated and / or optionally comprises one, two or three ring heteroatoms, where the heteroatoms are independently selected from O, N and S; (d ") C6-C12 aryl, where the aryl group is optionally substituted; (e") C6-C12 heteroaryl, where the heteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the heteroaryl group is optionally substituted; (f ") C7-C14 alkylaryl where the alkylaryl group is optionally substituted and / or where an alkyl part of the alkylaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S; and (g ") C7-C14 alkylheteroaryl, where the alkylheteroaryl group comprises one, two or three heteroatoms

Independently selected from O, N and S and / or where the alkylheteroaryl group is optionally substituted, and / or where an alkyl part of the alkylheteroaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one , two or three heteroatoms, where the heteroatoms are independently selected from O, N and S.

[0066] The CFG is preferably selected from the group consisting of -OH, -OR7, -NH2; -NH (R7), -N (R7) 2, -C (O) OR7, -C (O) R7, - C (O) NHR7, and -C (O) NR72, where R7 is as defined above for FG. For example, the FG can be -OH and the CFG can be -COOH.

[0067] The nanoparticles according to the general formulas (3), (4), (24) and (25) can be prepared by a process where a

PA polymer according to the general formulas (1), (2), (10) or (11), respectively, is reacted with a reagent according to formula (26a) or formula (26b):

FG-R6-NHR30 (26a)

FG-R6-SH (26b)

where FG and R6 are as defined above, FG is optionally hydrogen. Other useful FGs include biologically or pharmacologically active groups, for example oligo and polypeptides. Suitable examples for the reagents according to formula (26a) and (26b) are ethylenediamine, diethylenetriamine, triethylenetetramine,

Tetraethylenepentamine, 1,6-diamino hexane, PEG terminated amine, PPO terminated amine, PEG terminated thiol and PPO terminated thiol.

[0068] However, as will be apparent to those skilled in the art, the reagent according to formula (26b) may not only react with acrylate groups of PA polymers according to any of formulas (1), (2), ( 10) and (11), but also with the SS- groups (if any) in R2 of the PA polymer according to formula (1) or formula (2) or with the SS-group as represented by Z in the polymers PA according to formula (10) or formula (11). Additionally, since R3 of the PA polymer according to the formula (1) or the formula (2) and R30 of the PA polymer according to the formula (10) or the formula (11) can comprise functional groups, it can also be provided that the reagent according to Formula (26a) can react with these functional groups. In addition, instead of the reagent according to formula (26a), also reagents according to

The formula FG-R6-RG can be used, where RG is selected from the group consisting of -OH, -OR7, -C (O) OR, C (O) R7, -C (O) NHR7, -C (O ) NR72 and -SO2Cl, where R7 is as defined above, when R3 and R30 comprise for example a primary or secondary amino group.

The nanogel

[0069] The present invention also relates to a nanogel. The nanogels according to the present invention have as important advantages that they are stable in dissolved and dispersed form. The nanogels according to the present invention are also stable in storage. The solutions of the nanogels can be frozen without losing their integrity and can be lyophilized to a powder form that can easily be restored to a solution without loss of activity or

65 integrity.

image27

[0070] The nanogel is prepared by crosslinking the PA polymer according to general formulas (1) and (2), where the crosslinking is preferably performed by UV radiation, preferably UV radiation with a wavelength of approximately 365 nm. The crosslinking reaction is preferably performed in the presence of a photoinitiator. The crosslinking reaction is also preferably performed in a water-in-oil emulsion. It is also

It is preferred that the crosslinking reaction is carried out at a pH of less than 7, preferably less than about 6. Preferably, the pH is greater than about 1, preferably greater than about 2.

[0071] The present invention therefore also relates to a nanogel that is obtainable by the process described above comprising the cross-linking step of a PA polymer according to the general formulas (1), (2), (10) and ( 11), preferably subjecting the PA polymer to UV radiation.

Loaded Nanogeles

[0072] According to the invention, it is preferred that the nanogel further comprises a biologically active component selected from the group of RNA (in particular siRNA) or derivatives or fragments thereof, DNA or derivatives or fragments thereof, (oligo) peptides and derivatives thereof, and proteins and derivatives thereof.

[0073] The nanogel is charged by contacting the biologically active component with the nanogel in an aqueous solvent system, preferably a buffered aqueous solvent system, where the aqueous solvent system preferably has a physiological pH. Methods for loading nanogels are known in the art.

Surface modification of nanogels

[0074] The nanogel according to the present invention, either in the loaded form or in the unloaded form, can also be functionalized by post-modification.

[0075] According to a preferred embodiment of post-modification, the nanogel that can be obtained by crosslinking PA polymers according to formulas (1) or (2), where R3 is independently selected from the group consisting of:

(a1) substituted C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S; (b1) substituted C3-C12 cycloalkyl, where the cycloalkyl group is optionally (partially) unsaturated and / or

Optionally it comprises one, two or three ring heteroatoms, where the heteroatoms are independently selected from O, N and S; (c1) substituted C6-C12 aryl; (d1) C6-C12 substituted heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S; (e1) substituted C7-C14 alkylaryl, where an alkyl part of the alkylaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O , N and S; (f1) substituted C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where an alkyl part of the alkylheteroaryl group is linear

45 or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S;

where the above groups (a) - (f) comprise a functional group (FG) as a substituent that is capable of forming a covalent bond with a complementary functional group (CFG) of a reagent for post-modification R and where the nanogels optionally they comprise a biologically active component, it is reacted with said reagent R according to the process:

image28

[0076] The CFG group is capable of forming a covalent bond with the FG group. Examples suitable for groups FG and 55 CFG with well known by the person skilled in the art. For example, the FG can be a -COOH group while the CFG group is a -NH2 group.

[0077] According to a preferred embodiment, the functional group FG is selected from the group consisting of a group selected from -OH, -OR7, -NH2; -NH (R7), -N (R7) 2, -C (O) OR7, -C (O) R7, -C (O) NHR7 and -C (O) NR72, where R7 is independently selected from the group consisting in:

(a ") H; (b") C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms ,

65 where the heteroatoms are independently selected from O, N and S;

image29

(c ") C3-C12 cycloalkyl, where the cycloalkyl group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one, two or three ring heteroatoms, where the heteroatoms are independently selected from O, N and S; (d ") C6-C12 aryl, where the aryl group is optionally substituted;

5 (e ") C6-C12 heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the heteroaryl group is optionally substituted; (f") C7-C14 alkylaryl, wherein the alkylaryl group is optionally substituted and / or where an alkyl part of the alkylaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O , N and S; and (g ") C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroaryl group is optionally substituted, and / or where an alkyl part of the group alkylheteroaryl is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are

15 independently selected from O, N and S.

[0078] According to another preferred embodiment of the post-modification, the nanogel that can be obtained by crosslinking PA polymers according to formulas (10) or (11), is reacted with a post-modification reagent R 'according to the process:

image30

[0079] The groups R and R 'are preferably 2-thioethyl, 2-hydroxyethyl, and residues of PEG (polyethylene oxide) residues or residues of PPO (polypropylenoxide) having an average molecular weight in Mn number of about 500 to about 10,000 . As will be apparent to those skilled in the art, the R'-SH reagent may also

React with the S-S- groups (if any) in the nanogel as described above for PA polymers according to formulas (1), (2), (10) and formula (11).

[0080] The present invention therefore further relates to modified surface nanogels that are obtainable by a first process that includes the steps of:

(one)

crosslinking a PA polymer according to general formulas (1), (2), (10) or (11), preferably subjecting the PA polymer to UV radiation to form a nanogel;

(2)

optionally loading the nanogel with a biologically active component selected from the RNA group (in

particular siRNA) or derivatives or fragments thereof, DNA or derivatives or fragments thereof, (oligo) peptides and derivatives thereof, and proteins and derivatives thereof; Y

(3) react the nanogel with an R-CFG or R'-SH reagent.

[0081] Modified surface nanogels are also obtainable by a second process that includes the steps of:

(1 ') crosslinking a PA polymer according to general formulas (1), (2), (10) or (11), preferably subjecting the PA polymer to UV radiation to form a nanogel; (2 ') react the nanogel with an R-CFG or R'-SH reagent; and (3 ') optionally loading the nanogel with a biologically active component selected from the RNA group

45 (in particular siRNA) or derivatives or fragments thereof, DNA or derivatives or fragments thereof, (oligo) peptides and derivatives thereof, and proteins and derivatives thereof.

[0082] Modified surface nanogels are also obtainable by a third process that includes the steps of:

(1 ") reacting the nanogel with an R-CFG or R'-SH reagent thus forming a functional nanogel; (2") crosslinking the functionalized nanogel, preferably subjecting the functionalized nanogel to UV radiation to form a nanogel; and (3 ") optionally loading the nanogel with a biologically active component selected from the group of RNA (in particular siRNA) or derivatives or fragments thereof, DNA or derivatives or fragments thereof,

55 (oligo) peptides and derivatives thereof, and proteins and derivatives thereof.

[0083] In step (1) of the first process and stage (1 ') of the second process, not all acrylate groups need to be crosslinked so that uncrosslinked acrylate groups can be functionalized in step (3) of the first process and stage (2) of the second process, respectively. Also, in step (1 ") of the third process, not all acrylate groups need to be functionalized to those uncrosslinked acrylate groups can still be crosslinked. Therefore, the product obtained in any of these three processes can be very complex. In the present invention, it is even preferred that when a functionalization step is first performed, not all acrylate groups are functionalized thus allowing more crosslinking of the remaining acrylate groups, and it is also preferred when a crosslinking step is first performed not all groups Acrylate are crosslinked thus allowing more functionalization of the remaining acrylate groups .. One skilled in the art will be well able to select the

image31

appropriate reaction conditions, in particular by selecting appropriate molar ratios of reagents and raw materials, to control the degree of crosslinking and functionalization. In addition, and as described above, there are other reactive groups in the PA polymers, for example S-S- groups, which can be functionalized together with the acrylate groups.

5 Applications

[0084] The nanogels according to the present invention are very suitable as drug delivery carriers due to their high loading capacity, high stability and receptivity to environmental factors, such as ionic strength, reduction potential, pH and temperature. In particular, the nanogels according to the present invention have additional benefits for delivering biologically active components on self-assembled polymeric nanogranulose systems as delivery vehicles, since in the case of these nanogels all polymeric constituents are incorporated into the delivery nanogel while in the delivery self-assembled polymeric nanogranular systems normally the excess of free polymer in the solution is necessary due to the conditions of

15 balance. This frequently causes effects of unwanted cytotoxicity and in diluting the destabilization of the nanoparticle formulation.

Examples

20 Materials and methods

[0085] Cystamine bisacrylamide (CBA, Polysciences, USA) was purchased in the highest degree of purity and used without further purification. Low molecular weight polyethyleneimine (PEI) with weight average molecular weight 800 Da (PEI800) was obtained from Aldrich. An Igracure 2959 photoinitiator was purchased from Ciba. ABIL EM 90 surfactant was

25 purchased from Degussa-Goldschmidt (Evonik). SiRNA anti-luciferase was purchased from Eurogentec, AllStars. SiRNA of negative control was purchased from Qiagen. All other chemicals were purchased from Aldrich. All reagents and solvents were reagent grade and were used without further purification. NMR spectra were recorded in Varian Unity 300 (1H NMR 300 MHz) using tetramethylsilane (TMS) as an internal standard.

Example 1: synthesis of diacriloyl macromer (A- (CBA-ABOL) n-A).

[0086] Approximately 0.48 g (0.0054 mol.) Of 4-amino-1-butanol (ABOL) and 1.56 g (0.006 mol) of CBA (9/10 mol / mol ABOL / CBA ratio) are added to 3 ml of a solution of CaCl2 (0.4 M) in methanol-water 3/1 (v / v) and the reaction flask was sealed. The polymerization was carried out at 50 ° C and the reaction was allowed to proceed for 30 h, producing a

35 viscous solution. The reaction was monitored following with 1H-NMR, the integrated areas of two acryloyl peaks (5.55 and 6.05) and two methylene peaks of the butanol side groups (1.58 ppm and 1.78 ppm). At the end of the polymerization, NMR integrations indicated that the macromer (A- (CBA-ABOL) n-A) contained about six ABOL units per acryloyl group, that is n = 12.

40 Example 2: synthesis of hyperbranched p (CBA-ABOL / PEI)

[0087] A volume of 2.12, 1.06, 0.53 or 0.26 ml of a low molecular weight PEI 800 solution (100 mg / ml) was added to 1.34 g Ap macromer (CBA- ABOL) 12-A diacriloyl in 3 ml methanol-water 3/1 (v / v) to achieve a desired molar ratio of acryloyl to PEI groups of 3/1, 6/1, 12/1 and 24/1, respectively. The mixture was diluted with a 0.4 M solution of CaCl2 in methanol-water 3/1 (v / v) to a final PEI concentration of 10 mM to prevent gelation during the reaction. The reaction was carried out at 50 C for 24 h. Subsequently, one half of the reactive mixture containing encapsulated end of p (CBA-ABOL) / PEI with CBA acryloyl groups was separated for the synthesis of the nanogels. For the second half of the reaction mixture, a 10-fold excess of ethylenediamine (EDA) was added to transform all terminal acryl CBA groups into amines. The resulting solution of p (CBA

50 ABOL) / End PEI coated with ethylenediamine was diluted with water to approximately 30 ml, acidified with 4 M HCl at pH ~ 4, and then purified using an ultrafiltration membrane (MWCO 1000 g / mol). After lyophilization, the hyperbranched polymer (CBA-ABOL) / PEI terminated with EDA was collected as HCI-salt. Yield 30-50%. The ABOL / Acryloyl ratios for different than p (CBA-ABOL) / PEI are summarized in Table 1.

55 Table 1

p (CBA-ABOL) / PEI13%

p (CBA-ABOL) / PEI7% p (CBA-ABOL) / PEI4% p (CBA-ABOL) / PEI 2%

Sample No.

one 2 3 4

PEI content in feed,% by weight

13 7 4 2

Acryloyl / PEI (mol / mol) in the feed

3 6 12 24

ABOL / Acryloyl (mol / mol) inp (CBA-ABOL) / PEI remaining

> 100 30 18 13

image32

Example 3: synthesis of p (CBA-ABOL) / hyperbranched PAMAM

[0088] A volume of 1.16, 0.87 or 0.58 ml of a second generation PAMAM dendrimer solution (G2) (100 mg / ml) was added to 1.34 g Ap macromer (CBA-ABOL) ) 12-A diacriloyl in 3 ml methanol-water 3/1 (v / v) to achieve a desired molar ratio of acryloyl groups to PAMAM molecules of 10 / 1.15 / 1 and 20/1, respectively. The mixture was diluted by a 0.4M solution of CaCl2 in methanol-water 3/1 (vol./vol.) To a final PAMAM concentration of 10 mM to prevent gelation during the reaction. The reaction was carried out at 50 ° C for 24 h. Subsequently, one half of the reactive mixture containing p-coated end (CBA-ABOL) / PAMAM with CBA acryloyl groups was separated for the synthesis of nanogels. To the second half of the reaction mixture, a 10-fold excess of 10 ethylenediamine (EDA) was added to transform all terminal acryl CBA groups into amines. The resulting solution of p (CBA-ABOL) / ethylene diamine-coated end PAMAM was diluted with water to approximately 30 ml, acidified with 4 M HCl at pH ~ 4, and then purified using an ultrafiltration membrane (MWCO 1000 g / mol ). After lyophilization, the EDA-terminated hyperbranched p (CBA-ABOL) / PAMAM polymer was collected as HCI-salt. Yield 30-50%. The ABOL / Acryloyl ratios for different compositions of p (CBA

15 ABOL) / PAMAM are summarized in table 2

Table 2

p (CBA-ABOL) / PAMAM-8%

p (CBA-ABOL) / PAMAM-6% p (CBA-ABOL) / PAMAM-4%

Sample No.

5 6 7

PAMAM content in feed (% by weight)

8 6 4

Acryloyl / PAMAM in the feed (mol / mol)

10 fifteen twenty

ABOL / Acryloyl in p (CBA-ABOL) / PAMAM (mol / mol) remaining

2. 3 14 13

twenty

Example 4: synthesis of p (CBA-ABOL) / hyperbranched oligoamines terminated with EDA

[0089] EDA-terminated hyperbranched polymers were prepared by reacting the hyperbranched polymers of Tables 1 and 2 with an excess of EDA. The reactions were allowed to proceed until complete disappearance of acryloyl groups occurred, as confirmed by NMR.

Table 3

Hyperbranched polymer

p (CBA-ABOL) / PEI13% p (CBA-ABOL) / PEI7% p (CBA-ABOL) / PEI4% p (CBA-ABOL) / PEI -2%

Sample No.

one 2 3 4

Sample No. of hyperbranched polymer terminated with EDA

8 9 10 eleven

Example 5: synthesis of nanogels of p (CBA-ABOL) / oligoamine (NG-PAAs) [0090] The following hyperbranched polymers (see Table 3) were crosslinked as follows. Table 4

Hyperbranched polymer

p (CBA-ABOL) / PEI2% p (CBA-ABOL) / PEI4% p (CBA-ABOL) / PAMAM-6% p (CBA-ABOL) / PAMAM-4%

Sample No. of NG-PAA

12 13 14 fifteen

35

[0091] A solution of 500 mg p (CBA-ABOL) / oligoamine terminated with acryloyl (oligoamine = PEI or PAMAM) was acidified at room temperature with 4 M HCl at pH 4. Methanol was evaporated from the reaction solution in a flow of nitrogen. The volume of the solution was subsequently adjusted to 4 ml with deionized water. After the addition of 5 mg of Igracure 2959 photoinitiator, the aqueous phase was emulsified in 30 ml of mineral oil containing 10% ABIL EM 90 surfactant through ultrasound (Bandelin Sonoplus GM 2070) for 5 min (75% amplitude ). Immediately after ultrasound, the nanogotites were crosslinked by UV irradiation (5 mW / cm2 at 365 nm for 1 h) under stirring. To eliminate the continuous phase, the emulsion was diluted with 100 ml of 1/1 (v / v) acetone / hexane mixture. The nanogels of p (CBA-ABOL) / crosslinked oligoamine (with acronym NG-PAAs) were

image33

infragranulates by centrifugation (8000 r.p.m., 5 min), washed from the surfactant four times with acetone / hexane mixture and redispersed in 30 ml of deionized water. After purification using an ultrafiltration membrane (MWCO 10000 g / mol) and lyophilization, the NG-PAAs were stored at -20 ° C. The yields were 20-25%.

5

Example 6: synthesis of poly (ethylene glycol) is thiol-functionalized (PEG-SH)

[0092] Thiol-functionalized PEG (PEG-SH) with molecular weight of 2000 g / mol was synthesized from PEG methoxy in a three-step synthesis as described below.

PEG-mesylate

[0093] The PEG methoxy hydroxyl group was activated by mesylation according to a slightly modified procedure as published by Elbert et al. (D. L. Elbert, J. A. Hubbell, Biomacromolecules 2001, 2, 430). In a typical example

10.0 g (5 mmol, 1 eq) of PEG methoxy (Pm = 2000 g / mol) was dried twice in a 70 ml azeotropic distillation of dry toluene. After the second distillation step, the PEG was dissolved in 20 ml of dry dichloromethane followed by the addition of 6.6 ml (15 mmol, 3 eq) of trioctylamine. Subsequently, the solution was cooled in an ice bath and 1.72 g of mesyl chloride was added dropwise. The reaction was allowed to proceed overnight at room temperature under a nitrogen atmosphere, followed by precipitation in diethyl ether. PEG-mesylate was finally collected as a white powder by filtration and dried under vacuum. Yield: 83%. 1H NMR (CDCl3) δ (ppm): 3.08 (s, 3H, OSO2CH3); 3.37 (s, 3H, CH3OCH2); 3.40 (t, 2H, CH3OCH2); 3.52-3.90 (m, 176H, PEG); 4.38 (t, 2H, CH2CH2OSO2CH3).

PEG-thioacetate

[0094] In the second step the mesylate group was converted into a thioacetate according to a modified bibliography procedure (C. Woghiren, B. Sharma, S. Stein, Bioconjugate Chem. 1993, 4, 314). Therefore, 3.0 g (1.5 mmol, 1 eq) of PEG-mesylate was dissolved in 10 ml of dry pyridine and separately 2.23 g (19.5 mmol, 13 eq) of potassium thioacetate was dissolved in 28 ml of a 2/1 (v / v) mixture of pyridine / dry methanol. The solution of PEG-mesylate and 2.6 ml of trioctylamine was then added to the solution of potassium thioacetate and the reaction mixture was stirred overnight at room temperature under a nitrogen atmosphere. All solvents were evaporated, the residue was dissolved in 10 ml of saline solution and extracted four times with dichloromethane. The organic phase was dried magnesium sulfate, concentrated and precipitated twice in diethyl ether. PEG-thioacetate was finally collected as a white powder by filtration and dried under vacuum. Yield: 78%. 1H NMR (CDCl3) δ (ppm): 2.33 (s, 3H, CH2SCOCH3);

35 3.08 (t, 3H, CH2SCOCH3); 3.37 (s, 3H, CH3OCH2); 3.40 (t, 2H, CH3OCH2); 3.52 -3.90 (m, 176H, PEG).

PEG-thiol (PEG-SH)

[0095] In the last step the thioacetate was unprotected to produce PEG-SH (O. B. Wallace, D. M. Springer, Tetrahedron Lett. 1998, 39, 2693). Therefore, 2.3 g (1.2 mmol, 1 eq) of PEG-thioacetate was dissolved in 10 ml of methanol under a nitrogen atmosphere. To this solution 81 mg (1.2 mmol, 1 eq) of sodium thiomethoxide dissolved in 1 ml of methanol was added and the solution was stirred for 30 minutes at room temperature. Then the reaction mixture was poured into 20 ml of 0.1 M aqueous hydrochloric acid and extracted with dichloromethane. The organic layer was washed with saline solution and all solvents were removed by rotational evaporation. To delete the latest

45 impurities, PEG was dissolved in 40 ml of demineralized water containing 0.35 g of DTT (2.3 mmol, 2 eq) to reduce possibly oxidized and dialyzed ultrafiltration thiols with a molecular weight cut of 1000 g / mol. PEG-SH was finally obtained as a white spongy powder by lyophilization. Yield: 85%. 1H NMR (CDCl3) δ (ppm): 1.59 (t, 1H, CH2CH2SH); 2.68 (q, 2H, CH2CH2SH); 3.37 (s, 3H, CH3OCH2; 3.40 (t, 2H, CH3OCH2); 3.52-3.90 (m, 176H, PEG).

[0096] The total conversion of hydroxyl to thiol was determined by Ellman's reagent and found to be 100%.

Example 7: preparation of siRNA p (CBA-ABOL) / hyperbranched / oligoamine polyplejos

[0097] Multiples were prepared from CBA-ABOL (reference, example 1), sample numbers 1-4 (see, Table 1) and 5 and 6 (see, Table 2).

[0098] In the typical experiment, a siRNA solution was prepared at a final concentration of 0.025 mg / ml in glucose buffered with HEPES (HBG) (pH 7.4). A stock solution of p (CBA-ABOL) / hyperbranched oligoamine (1.25 mg / ml) in HBG (pH 7.4) was prepared and was used for the preparation of polyplejos at 50/1 p / p polymer ratio / SiRNA. This solution was repeatedly diluted 1: 1 with HBG to prepare multiples with 25/1, 12/1 and 6/1 w / w polymer / siRNA ratio. Typically equal volumes of siRNA and solutions of p (CBA-ABOL) / p oligoamine (CBA-ABOL) / hyperbranched oligoamine were mixed in a 1 ml Eppendorf tube. After mixing, the multiplex solution was incubated for 20 min at room temperature. For higher proportions (or doses

65 applied) increased concentrations of the components were applied.

image34

Example 8: NG-PAA nanoge siRNA loading

[0099] The nanogels of Example 4 as well as a nanogel obtained from the diacriloyl macromer (A- (CBA5 ABOL) n-A) of Example 1 were loaded with siRNA.

[0100] In the typical experiment, a siRNA solution was prepared at a final concentration of 0.025 mg / ml in glucose buffered with HEPES (HBG) (pH 7.4). A dispersion of NG-PAAs (1.25 mg / ml) in HBG (pH 7.4) was prepared and used for loading at 50/1 weight ratio nanogel / siRNA. The NG-PAA dispersion was repeatedly diluted 1: 1 with HBG to prepare nanogels loaded with siRNA with 25/1, 12/1 and 6/1 weight ratio of polymer / siRNA. Typically equal volumes of siRNA and nanogel solutions were mixed in a 1 ml Eppendorf tube and incubated for 20 min at room temperature. For higher proportions (or applied doses) higher concentrations of the appropriate components were applied in the same volumes as given above.

fifteen

Example 9: ethidium bromide (EtBr) displacement test

[0101] The ability of the EDA-terminated hyperbranched polymers of example 4 and the NG-PAA nanogels of example 5 to condense siRNA was studied using an ethidium bromide (EtBr) assay. The polymer of p (CBA-ABOL) (example 1) was used as a reference.

[0102] A solution of the hyperbranched polymer or NG-PAA nanogel was added step by step to the siRNA solution (10 µg / mL) in HBG containing EtBr (0.4 µg / mL). After each step, the fluorescence intensity was monitored (λex = 510 nm, λem = 590 nm). The fluorescence intensity of the EtBr solution in the presence of

25 Free siRNA corresponded to 0% condensation, while fluorescence intensity without siRNA corresponded to 100% DNA condensation.

[0103] The results are shown in Figures 1-3. Figure 1 shows that the polymer p (CBA-ABOL) itself has a rather low efficiency in the complexation of siRNA. However, hyperbranched polymers terminated with EDA and NG-PAA nanogels have a significantly higher binding capacity. Hyperbranched polymers terminated with EDA and NG-PAA nanogels containing PEI showed higher affinity for siRNA than those containing PAMAM (Figures 1 and 2). NG-PAA nanogels have a higher capacity to condense siRNA than the corresponding EDA-terminated hyperbranched polymers (Figure 3).

35 Example 10: in vitro gene silencing

[0104] The drop in efficiency was determined by silencing the expression of luciferase in NCI-H1299 cells, stably expressing firefly luciferase (donated by Prof. G. Storm, University of Utrecht, The Netherlands). The drop and cell viability were evaluated in two parallel sessions, using antiluciferase and non-coding siRNA, respectively. The cells were seeded in 96-well plates with a density of 8,000 cells per well. After 24 h of incubation at 37 ° C in a humidified atmosphere containing 5% CO2, the medium was replaced with 100 µl medium without fresh serum. Both anti-luciferase siRNA and non-coding siRNA were used. Lipofectamine 2000 (LF) was used as a reference and complexes were prepared according to the manufacturer's protocol. Multiflexes or nanogels loaded with siRNA (10 µl per well) were added to the cells in triplicate after

One hour of incubation with fresh medium, resulting in a final siRNA concentration of 72 nM. After two hours of transfection in the serum-free medium, the polyplex medium was replaced by the complete culture medium and the cells were incubated for another 48 h. The cells were lysed in 50 µl of lysis buffer and 20 µl of the cell lysate was mixed with 50 µl of luciferase assay reagent containing the substrate luciferin. After 100-220 seconds (in this period the emitted light is constant) the luciferase activity was determined by measuring the luminescence at 25 ° C for 4 seconds in a PerkinElmer 1420 Victor3 plate reader. Luciferase activity of untreated cells was defined as 100% expression.

[0105] Figures 4-6 show the gene that silences the efficiency of samples number 1-4 according to example 2 (figure 4), samples number 5 and 6 according to example 3 (figure 5) and nanogels NG-PAA according to example 5

55 (figure 6). The numbers on the x-axis represent the ratio of polymer / siRNA. The CBA / ABOL polymer showed no efficiency in the delivery of siRNA.

Example 11: measurement of particle size and zeta potential.

[0106] The particle sizes and zeta potentials of the charged and discharged multiples and the nanogels were measured by laser light scattering using a Zetasizer Nano ZS (Malvern, UK).

[0107] At high proportions of polymer / siRNA mixture (w / w 12 or more) no significant differences in size of the polyplex particles formed by hyperbranched polymers terminated with different EDA were observed. All 65 EDA-terminated hyperbranched polymers formed small multiples with sizes ranging from about 80 to about 120 nm and zeta potentials were about 30 to

image35

5 [0108] The loading of nanogels led to deflation and gradual reduction of their sizes after the reduction of a mixing ratio. Sample size # 12 decreased from approximately 120 nm (discharged) to approximately 90 nm (loaded w / w 50) to approximately 75 nm (loaded w / w 12).

[0109] Another reduction in the mixing ratio (increase in siRNA load) led to the aggregation of

10 nanogel particles similarly were observed for hyperbranched polymers terminated with EDA. The zeta potentials of the nanogels dropped slightly after loading. For sample 12, the zeta potential decreased from approximately 28 mV (discharged) to approximately 26 mV (loaded p / p 50) to approximately 20 mV (loaded p / p 12).

15 Example 12: cytotoxicity

[0110] Cell viability studies were performed with NCI-H1299 cells in the absence and in the presence of 10% FBS. The cells were seeded in 96-well plates with a density of 8,000 cells per well 24 h before transfection. A dilution series of hyperbranched polymers or NG-PAA nanogel solutions were prepared from 12.5 to 0.02 mg / ml and 10 µl of the solution was added to 100 µl of growth medium. After 24 hours of incubation, cell viability was measured using an XTT assay, where the XTT value for untreated cells (cells not exposed to transfection systems) was taken as 100% cell viability. The cell viability curves for samples number 4 and 12 in the absence of serum are shown in Figure 7. It is shown that especially the nanogels show the lowest cytotoxicity. By comparison: the polymer concentration is 0.055

25 mg / mL in cellular transfections at polymer / siRNA ratio 48/1.

Example 13: In vitro transfection under serum conditions

[0111] Transfection studies were performed with NCI-H1299 cells according to a similar procedure as

30 described in Example 10. However, in this case, the transfection medium also contained 10% FBS and the cells were incubated with siRNA-loaded nanogels for 48 h before the luciferase assay was performed. Sample solutions of nanogel loaded with siRNA sample No. 12 with increasing dose of siRNA (resulting in 125, 250 and 500 ng of siRNA per well (100 µL) in the incubation medium, ie 72, 144 and 288 nM, respectively), or solutions with constant dose of siRNA (72 nM) with increasing proportion of nanogel

Sample No. 12 was prepared according to the procedure described in Example 8. The results of luciferase gene silencing for these different formulations are shown in Figures 8 and 9, respectively.

Example 14: surface modification of NG-PAA nanogel formulations

[0112] The surface of sample 12 was modified by PEG using an exchange reaction between terminal -SH in the PEG chains and group -S-S-in the nanogels. In the typical experiment, the siRNA solution (0.05 mg / ml) was mixed with an equal volume of nanogel solution of 2.5 mg / ml in HBG (pH 7.4). After mixing, the nanogel solution was incubated for 20 min at room temperature and an equal volume of the PEG-SH solution in HBG was added. The concentration of PEG-SH varied from 0.125 mg / ml to 2.5 mg / ml to achieve proportions of

45 PEG-SH / nanogel from 0.1 to 2. The sizes and zeta potentials of the nanoparticles were subsequently followed by DLS. For studies of colloid stabilities a defined volume of the NaCl solution (3.15 M) was added 40 min after the addition of PEG-SH to achieve a final concentration of 150 mM NaCl. After this, the particle sizes were monitored as a function of time. Results for size and zeta potential are shown in Table 4 before and after surface modification by PEG 2 kDa.

50 Table 5

10% p / p PEG

20% p / p PEG 40% p / p PEG 100% p / p PEG

0 min

40 min  0 min 40 min  0 min 40 min  0 min 40 min

Size, nm

91 106 95 109 92 112 94 106

Zeta, mV

26 twenty-one 26 twenty-one 26 10.6 27 8.9

[0113] Figure 10 shows data for delivery of siRNA to H1299 cells by sample No. 12 modified with PEG. 55 Proportion of polymer / siRNA 50 p / p. Serum free conditions.

Example 15: stability of nanogel formulations against freeze-thaw and lyophilization cycles

image36

[0114] Properties formulations with siRNA of hyperbranched sample No. 4 and nanogel sample No. 12 after the freeze-thaw and lyophilization cycles were studied by DLS.

[0115] Freezing of the formulations of sample No. 4 resulted in the aggregation of nanoparticles of

5 multiplex. The size of the multiples at a mixing ratio of 50 w / w increased from about 80 nm to about 600 nm after a freeze-thaw cycle. It was also not possible to reconstruct the lyophilized formulation by adding MilliQ water. After 15 min of vortexing, a significant amount of insoluble material was still present in the solution.

[0116] In the case of the nanogel formulations of sample 12, however, virtually no change in particle size was observed after both freeze-thaw and lyophilization. The particle sizes at 50 w / w mixing ratio of the initial formulation and those after a freeze-thaw cycle and after lyophilization were approximately 91, approximately 94 and approximately 103 nm, respectively. The formulations did not lose their activity after these processes

15 (Figure 11: the gray bars on the left represent the anti-LUC siRNA formulation, the gray bar on the right is senseless siRNA as a control. Polymer / siRNA ratio 50 w / w. Serum free conditions. The three Formulations showed the same efficiency in the fall of the target gene.

Claims (16)

image 1  Claims 1. Polymer according to formulas (1) and (2): image2 where: 10 A is independently selected from a simple direct carbon-carbon bond (ie a structure where A is absent), O, N (R1) and S; R1 is independently selected from H and CH3; R2 is independently selected from the group consisting of: 15 (a) C1-C40 alkylene, where the alkylene group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where the alkylene group is interrupted by one or more groups -SS-; (b) C3-C40 cycloalkylene, where the cycloalkylene group is optionally substituted and / or is optionally (Partially) unsaturated and / or optionally comprises one or more heteroatoms in the ring, where the heteroatoms are independently selected from O, N and S, and / or where the cycloalkylene group is interrupted by one or more SS-groups outside the ring ; (c) C6-C40 arylene, where the arylene group is optionally substituted; (d) C6-C40 heteroarylene, where the heteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the heteroarylene group is optionally substituted; (e) C7-C40 alkylarylene, where the alkylarylene group is optionally substituted and / or where an alkyl part of the alkylarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where an alkyl part of the alkylarylene group is interrupted by one 30 or several groups -S-S-; (f) C7-C40 alkylheteroarylene, where the alkylheteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroarylene group is optionally substituted, and / or where an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, Where the heteroatoms are independently selected from O, N and S, and / or where an alkyl part of the alkylheteroarylene group is interrupted by one or more groups -S-S-; Y (g) a group where two C7-C40 (hetero) arylene groups and / or C7-C40 alkyl (hetero) arylene groups are connected to each other by a group -SS-, where the alkyl part of the (hetero) arylene alkyl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more 40 heteroatoms, where the heteroatoms are independently selected from O, N and S; R3 is independently selected from the group consisting of:

(to)

H;

(b)

C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S;

(C)

C3-C12 cycloalkyl, where the cycloalkyl group is optionally substituted and / or is optionally

image3 5 (partially) unsaturated and / or optionally comprises one, two or three ring heteroatoms, where the heteroatoms are independently selected from O, N and S;

(d)

C6-C12 aryl, where the aryl group is optionally substituted;

(and)

C6-C12 heteroaryl, where the heteroaryl group comprises one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S, and where the heteroaryl group is optionally substituted;

(F)

C7-C14 alkylaryl, where the alkylaryl group is optionally substituted and / or where an alkyl part of the alkylaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S; Y

15 (g) C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroaryl group is optionally substituted, and / or where an alkyl part of the alkylheteroaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S; R4 is independently selected from the group consisting of: (a) H; (b) C1-C10 alkyl, where the alkyl group can be linear or branched; 25 (c) C3-C12 cycloalkyl;

(d)

C6-C14 aryl;

(and)

C6-C14 heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S;

(F)

C7-C14 alkylaryl; Y

(g)

C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S;

R5 is independently selected from the group consisting of: (A) C1-C12 alkylene, where the alkylene group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S:

(b)

C3-C12 cycloalkylene, where the cycloalkylene group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one or more ring heteroatoms, where the heteroatoms are independently selected from O, N and S;

(C)

C6-C12 arylene, where the arylene group is optionally substituted;

(d)

C6-C12 heteroarylene, where the heteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the heteroarylene group is optionally substituted;

(and)

C7-C12 alkylarylene, where the alkylarylene group is optionally substituted and / or where a part of

The alkyl of the alkylarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S; Y (f) C7-C12 alkylheteroarylene, where the alkylheteroarylene group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroarylene group is optionally substituted, and / or where an alkyl part of the alkylheteroarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S; POL represents a polymeric core having a weight average molecular weight Pm of about 300 to about 25,000; a = 2-64; Y b = 1 -50.

2.

Polymer according to claim 1, wherein the weight average molecular weight Pm of the polymer polymer core is from about 300 to about 5,000.

3.

Polymer according to claim 1 or claim 2, wherein the polymer core POL is based on or selected

of a branched, hyperbranched, multi-arm, dentritic or star-type (co) polymer. 65

Four.

Polymer according to claim 3, wherein the (co) polymer has 2-64 terminal amino groups.

5.

Polymer according to claim 4, wherein the amino groups are primary amino groups.

6.

Polymer according to any of claims 1-5, wherein the POL polymer core is based on or selected from the group consisting of PEI, PAMAM, PPI, PEAN and PEAC.

7.

Polymer according to claim 6, wherein the PEI is represented by the general formulas (5a) and (5b):

image4 image5 where n is so that the PEI has a weight average molecular weight Mn of about 600 to about 3,000. fifteen 8. Polymer according to claim 6, wherein the POL polymer core is based on or selected from the group consisting of polymers represented by the general formulas (6) - (9): N (R8) 3-n [(CR92) m-N (R10R11)] n (6) 20 [(R10R11) N- (CR92) m] 2N-P-N [(CR92) m-N (R10R11)] 2 (7) N (R8) 3-n [(CR92) m-C (O) N (R9) - (CR92) m-N (R12R13)] n (8) 25 [(R12R13) N- (CR92) m-N (R9) C (O) - (CR92) m] 2N-P-N [(CR92) -C (O) NH- (CR92) m-N (R12R13)] 2 (9) where: R8 is a hydrogen atom, a linear or branched C1-C20 alkyl group or a group - [(CR142) q-X] or-R15, where X 30 is O or N (R8); m is 2, 3 or 4; image6 n is 2 or 3; o is 1-10; q is 2, 3 or 4; P is - (CR92) m-, a C6-C12 arylene group, a C6-C12 cycloalkylene group or a group [(CR142) q-X] p-C (R14) 2] -, where 5 X isOoN (R8) ypes1-10; R9 is a hydrogen atom or a linear or branched C1-C6 alkyl group; R10 and R11 are independently a hydrogen atom, a linear or branched C1-C6 alkyl group or a group of the formula - (CR142) qNR16R17, provided that R10 and R11 are not both a linear or branched C1-C6 alkyl group; R16 and R17 are independently a hydrogen atom, a linear or branched C1-C6 alkyl group or a group 10 of the formula - (CR142) qNR18R19, provided that R16 and R17 are not both a linear or branched C1-C6 alkyl group; R18 and R19 are independently a hydrogen atom, a linear or branched C1-C6 alkyl group or a group of the formula - (CR142) qNR20R21, provided that R18 and R19 are not both a linear or branched C1-C6 alkyl group; R20 and R21 are independently a hydrogen atom or a linear or branched C1-C6 alkyl group, provided that R20 and R21 are not both a linear or branched C1-C6 alkyl group; R22 is a hydrogen atom or a methyl group, provided that at least one R22 is a hydrogen atom; R15 is a hydrogen or a linear or branched C1-C20 alkyl group or a group - [(CR142) q-X] or-R15 as defined above; R12 and R13 are independently a hydrogen atom, a linear or branched C1-C6 alkyl group or a group of the formula - (CR92) mC (O) NH- (CR92) mN (R23R24), provided that R12 and R13 are not both a C1-C6 alkyl group 20 linear or branched; R23 and R24 are independently a hydrogen atom, a linear or branched C1-C6 alkyl group or a group of the formula - (CR92) mC (O) NH- (CR92) mN (R25R26), provided that R21 and R22 are not both a linear or branched C1-C6 alkyl group; R25 and R26 are independently a hydrogen atom, a linear or branched C1-C6 alkyl group or a group 25 of the formula - (CR92) m-C (O) NH- (CR92) m-N (R27R28), provided that R25 and R26 are not both a linear or branched C1-C6 alkyl group; R27 and R28 are independently a hydrogen atom or a linear or branched C1-C6 alkyl group, provided that R27 and R28 are not both a linear or branched C1-C6 alkyl group. 30 9. Process for the preparation of a PA polymer according to the general formulas (1) or (2), said process includes the steps of: (1) a monomer (I) according to the general formula (12): image7 Wherein R1, A and R2 are as defined in claim 1, it is reacted with a monomer (II) according to the general formula (13) or a monomer (III) according to the general formula (14): (H2N) -R3 (13) 40 HR3N-R5-NR3H (14) wherein R3 and R5 are as defined in claim 1, in a molar ratio of monomer (I): monomer (II) from about 1.5: 1 to about 10: 1 to form a macromer according to the general formulas (15) or (16): image8 Wherein b is as defined in claim 1; Y (2) reacting the macromer according to the general formulas (15) or (16) with a polymer according to the formula (17); [(R42) N] a-POL (17) wherein a and R4 are as defined in claim 1 and where at least one R4 is H. 10. PA polymer according to the general formulas (1) or (2) obtainable by the process according to claim 9. 15

eleven.

Nanogel comprising the PA polymer according to any one of claims 1-8 or 10.

12.

Nanogel according to claim 11, wherein the nanogel comprises a biologically active component.

13. Nanogel according to claim 12, wherein the biologically active component is selected from the group of RNA (in particular siRNA and mRNA) or derivatives or fragments thereof, DNA or derivatives or fragments thereof, (oligo) peptides and derivatives thereof. themselves, and proteins and derivatives thereof.

14.

Process for the preparation of the nanogel according to claim 11, wherein a PA polymer according to the general formulas (1) or (2) is crosslinked.

15. Process according to claim 14, wherein the cross-linking is carried out by UV radiation.

16.

Nanoparticle of the general formulas (3) and (4): 30

image9 image10 wherein R1, R2, R3, R4, R5, A, POL, a and b are as defined in claim 1; R6 is selected from the group consisting of: 5 (a) C1-C40 alkylene, where the alkylene group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O , N and S, and / or where the alkylene group is interrupted by one or more groups -SS-; or 10 (b) C3-C40 cycloalkylene, where the cycloalkylene group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one or more ring heteroatoms, where the heteroatoms are independently selected from O, N and S , and / or where the cycloalkylene group is interrupted by one or more SS-groups outside the ring; Y FG is a functional group that is selected from the group consisting of -OH, -OR7, -NH2; -NH (R7), -N (R7) 2, C (O) OR7, -C (O) R7, -C (O) NHR7 and -C (O) NR72 where R7 is independently selected from the group consisting of: (a ") H; 20 (b ") C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently select O, N and S; (c ") C3-C12 cycloalkyl, where the cycloalkyl group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one, two or three ring heteroatoms, where the 25 heteroatoms are independently selected from O, N and S; (d ") C6-C12 aryl, where the aryl group is optionally substituted; (e") C6-C12 heteroaryl, where the heteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the heteroaryl group is optionally substituted; (f ") C7-C14 alkylaryl, where the alkylaryl group is optionally substituted and / or where an alkyl part 30 of the alkylaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S; and (g ") C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroaryl group is optionally Substituted, and / or where an alkyl part of the alkylheteroaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S. 17. Process for the preparation of a nanoparticle comprising the step of reacting a PA polymer according to general formulas (1) or (2) with a reagent according to formulas (26a) or (26b): FG-R6-NHR30 (26a) FG-R6-SH (26b) 45 R30 wherein R6 and FG are as defined in claim 16, FG is optionally H, and is independently selected from the group consisting of: (a) H; 50 (b) C1-C10 alkyl, where the alkyl group can be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are selected regardless of O, N and S; (c) C3-C12 cycloalkyl, where the cycloalkyl group is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one, two or three heteroatoms in the ring, where the heteroatoms are independently selected from O, N and S;

(d)

C6-C12 aryl, where the aryl group is optionally substituted;

(and)

C6-C12 heteroaryl, wherein the heteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the heteroaryl group is optionally substituted;

(F)

C7-C14 alkylaryl, where the alkylaryl group is optionally substituted and / or where an alkyl part of the alkylaryl group is linear or branched and is optionally (partially) unsaturated and / or is optionally

image11 5 interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S; Y (g) C7-C14 alkylheteroaryl, wherein the alkylheteroaryl group comprises one, two or three heteroatoms independently selected from O, N and S and / or where the alkylheteroaryl group is optionally substituted, and / or where an alkyl part of the alkylheteroaryl group is linear or branched and is optionally (partially) 10 unsaturated and / or is optionally interrupted by one, two or three heteroatoms, where the heteroatoms are independently selected from O, N and S. 18. Modified surface nanogel obtainable by a process that includes the steps of: 15 (1) crosslinking a PA polymer according to general formulas (1) or (2);

(2)

optionally loading the nanogel with a biologically active component, preferably selected from the group of RNA (in particular siRNA) or derivatives or fragments thereof, DNA or derivatives or fragments thereof, (oligo) peptides and derivatives thereof, and proteins and derivatives from the same; Y

(3)

react the nanogel with an R-CFG or R'-SH reagent, where:

R and R 'are selected from the group consisting of 2-thioethyl, 2-hydroxyethyl, PEG (polyethylene oxide) residues and PPO (polypropylene oxide) residues, where PEG residues and PPO residues have an average molecular weight in Mn number from about 500 to about 10,000; CFG is a group capable of forming a covalent bond with an FG group, where FG is as defined 25 in claim 16; and where the sequence of steps (1), (2) and (3) is: (1) - (2) - (3),. (1) - (3) - (2), or (3) - (1) - (2).